The velvet gene product does not release

We investigated a molecular connection between csnD and veA. A dependency of csnD and veA transcripts was analysed in Northern hybridisation experiments.

Wild-type, csnD deletion and veA1 strains were grown in liquid overnight culture for 18 h to synchronise the mycelia at the stage of developmental competence. This cell material was transferred to solid medium and induced asexually or sexually in light or in the dark on sealed plates. On the transcriptional level (Fig. 4.9), specific

csnD and veA RNA signals from this cell material, compared to rRNA, were not significantly altered after shift from vegetative to differentiating cultures in the wild-type strain and the veA1 strain. Apparently, the mean veA transcript levels were generally higher in the veA1 strain. csnD transcripts were present independent of the veA allelic state. Vice versa, the quantity of veA transcript was similar in csnD wild-type, deletion and overexpression strains. Taken together, transcription of csnD and veA proceeds independently, irrespective of the developmental state of the culture.

The phenotypic consequence of the veA1 deletion and the overproduction of the veA wild-type gene in a csnD deletion strain was analysed to test their genetic relationship. The ∆csnD/veA1 mutant (AGB192) showed both red pigmentation and aberrant cell forms like the csnD/veA+ strain (AGB195). AGB192 also produced Hülle cells and primordia that never matured to cleistothecia, though apparently less than the corresponding csnD/veA+ strain. (Fig. 4.8). Thus, the veA1 mutant i s unable to suppress the block in development caused by csnD deletion. All phenotypes of the deletion mutant AGB192 were complemented by ectopic integration of the corresponding csnD genomic fragment in strain AGB193. The A. nidulans ∆csnD/PniiA:veA strain (AGB220) showed red colouring, highly branched hyphae with aberrant cells were readily visible in this mutant, and also sexual development was blocked at the level of cleistothecial primordia (Fig. 4.8).

This indicates that even overproduction of VEA, which normally leads to enhanced cleistothecia production, does not lead to development of mature cleistothecia in a csnD deletion strain. Thus, neither high VEA levels nor the changed veA gene product are able to overrule the developmental block in csnD deletion strains.

Consequently, csnD is epistatic to the veA1 loss of function and veA gain of function mutations, which places this function of CSND in respect to specific sexual development genetically downstream of VEA.

v a s v a s

veA veA1 veA veA1

∆csnD csnD

csnD

veA

csnD ∆csnD

v a s v a s

rRNA

Fig. 4.9: Transcription of csnD and veA during development. A. nidulans strains AGB160 (csnD, veA), AGB162 (csnD, veA1), AGB195 (∆csnD, veA) and AGB192 (∆csnD, veA1) were pre-grown in submerged liquid culture, the developmentally competent mycelia were transferred to solid medium and induced either asexually in the light or sexually in the dark on sealed plates. Vegetative (v), asexual (a) and sexual (s) tissue types were harvested for RNA isolation for Northern hybridisation experiments using csnD and veA as specific probes. Levels of specific csnD and veA mRNAs were independent of the strain and the developmental conditions.

4.5 Discussion

This study identifies the existence of the COP9 signalosome in filamentous fungi and describes it as key regulator of fungal development. The csnD and csnE genes encode the fourth and fifth CSN subunits of A. nidulans. The deduced peptide sequences for CSND and CSNE contain PCI and MPN motifs, respectively, which are characteristic for proteins of the 26S proteasome lid, eIF3 and the COP9 signalosome multiprotein complexes (Kim et al. 2001). Protein-protein interactions between CSND and CSNE are comparable to that described for subunits 4 and 5 of the COP9 signalosome in other organisms (Kapelari et al. 2000; Tsuge et al.

2001). At the transcriptional level, csnD mRNAs are abundant in both vegetative and developing cultures. In analogy to this, specific mRNAs of CSN subunits were detected in all mouse embryonic and adult tissue tested (Bounpheng et al. 2000). A fusion of CSND to the green fluorescent protein is dispersed in the cytoplasm and clearly enriched in the nuclei. This is in agreement with observations in other organisms, where subunits of the CSN are predominantly localised in the nucleus as multiprotein complex, and subunits 4 to 8 were additionally found in the cytoplasm probably forming a smaller subcomplex (Chamovitz et al. 1996; Kwok et al. 1998; Tsuge et al. 2001; Tomoda et al. 2002). All our data suggest that the products of the two identified genes represent the first members of the COP9 signalosome in filamentous fungi.

In higher eukaryotes, defects in subunits of the COP9 signalosome result in severe developmental phenotypes and post-embryonic lethality (Wei et al. 1994;

Freilich et al. 1999), whereas malfunction of the complex in S. pombe is not lethal and leads to minor mutant phenotypes like delayed progression through the cell cycle and increased sensitivity to ultraviolet light (Mundt et al. 1999; Mundt et al.

2002). This work identified the COP9 signalosome of A. nidulans as a key regulator in the development of the organism, essential for proper regulation of metabolism, cell morphology, hyphal polarity, light-regulation and sexual reproduction (Fig. 4.10). The study of the COP9 signalosome in the model organism A. nidulans has three major advantages: it is easily accessible to molecular manipulations. In contrast to COP9 signalosome defects in higher eukaryotes, an A. nidulans strain defective in its sexual cycle is still viable and can propagate via its asexual cycle.

Last but not least, A. nidulans is evolutionary closer related to humans than plants are.

Malfunction of the COP9 signalosome in A. nidulans results in changes of secondary metabolism which is visible with the naked eye: overproduction of a red pigment. To date, we have no indication about the origin of this red substance. In A. nidulans, knowledge about regulation of secondary metabolism, and especially about red pigments, is rather restricted. Wild-type strains deposit brownish melanin in walls of older hyphae when mycelia are grown in submerged liquid culture (Pirt and Rowley 1969). This phenomenon is not altered in the csn deletion mutants compared to the wild-type (not shown). Notably, absence of melanin i s correlated with defective sexual reproduction in A. nidulans, indicating a cross-connection between secondary metabolism and sexual development (Champe et

al. 1994). The second known red pigment produced by A. nidulans is responsible for the colour of the ascospores: the anthraquinone asperthecin, which is difficult to isolate (Howard and Raistrick 1955). It seems striking that the red colouring in the csnD mutant becomes visible after two-three days of growth which is the time scale where also the first structures of sexual development become visible. Notably, CSN mutants of the plant A. thaliana overproduce anthocyan, the flowering colour (Misera et al. 1994). This raises the question whether the red pigment produced in the csn deletion strains is related to the one that dyes the ascospores. Preliminary tests using 0.5 M NaHCO3 and Na2CO3 according to Howard and Raistrick (1955) did not reveal whether the red colour of the csn deletion mutants was asperthecin (data not shown). The aberrant colouring of hyphae in the csn deletion strains appears independent of developmental induction by light and the allelic veA state.

Regulation of the production of the putative red pigment thus seems to be mainly mediated by time and/or growth phase. Thus, an impact of the COP9 signalosome in the internal regulation of onset of secondary metabolism and/or development i s conceivable. Astonishingly, red coloured hyphae are also seen in a veA deletion strain, but it is not clear whether the red pigment produced by ∆csn and ∆veA strains is identical.

The COP9 signalosome of A. nidulans is involved in the control of polar apical growth and lateral branching in surface-grown cultures. Generally, establishment of polar growth after germination and its later maintenance seem independent processes in A. nidulans, with a proposed persistent signal for ongoing apical extension (Momany et al. 1999). Young hyphae of surface-grown csn deletion colonies as well as hyphae grown in submerged liquid culture show

polarity

primordium

cleistothecium light signal

development maintenance

pigment

establishment primary metab.

CSND CSNE light

Fig. 4.10: Multifaceted role of the COP9 signalosome (CSN) in A. nidulans.

Protein-protein interaction between the two CSN subunits CSND and CSNE are indicated by paralleled bars. The impact of the COP9 signalosome in a light-dependent signalling pathway is indicated by a broken arrow line and the function of the CSN in several downstream target pathways by full arrow lines.

initiation of development secondary

metabolism

COP9 signalosome

sexual propagation

no obvious aberrance. Thus, the establishment of polarity seems not generally disturbed in the csn deletion strains. But maintenance of apical extension in elderly, surface-grown cell material seems to be a target of the COP9 signalosome. In A. nidulans wild-type strains, polarised apical growth is generally turned off during developmental programs. In the asexual propagation cycle, a switch from polarised growth to a bud-like growth form is seen during sterigmata formation and conidiation (Adams et al. 1998). As for propagation of sexual spores, ascogenous hyphae are formed that can be seen in young immature cleistothecia as branched filaments with knobby cells (Braus et al. 2002). A. nidulans strains with overproduction of the transcription factor STEA block vegetative growth and produce highly branched hyphae with small, knobby cells very similar to ascogenous tissue, though a direct relation has not been proven yet (Vallim et al. 2000). Nevertheless, the short and highly branched hyphae in elderly csn deletion strains do not morphologically resemble the phenotype described for the STEA overproduction strain and young ascogenous tissue. Similar to the polarity defect of A. nidulans csn deletion strains, malfunction of the A. thaliana COP9 signalosome causes aberrant cell morphologies. Transgenic plants with reduced CSN levels show a general increase in secondary inflorescences and a reduction of internode length and cell size. These phenomena are primarily due to a loss of apical dominance, which in turn is driven by the phytohormone auxin. In A. thaliana, the auxin-response is controlled by the COP9 signalosome, probably by degradation of the AUX/IAA transcriptional repressors (Schwechheimer et al. 2001). Auxin, a tryptophan-related hormone-like signal molecule, is product of secondary metabolism. We have recently reported a role of auxin for development in A. nidulans (Eckert et al. 2000). Strains auxotrophic for tryptophan arrest sexual development at the level of micro-cleistothecia, which is one step beyond the arrest at primordia seen in csn deletion strains. External supply of high amounts of tryptophan or traces of auxin released this developmental block. Future studies will focus on a possible co-ordination of sexual development and hormone signalling by the A. nidulans COP9 signalosome.

The severe mutant phenotype of csnD or csnE deletion in A. nidulans is a block in sexual development at the level of cleistothecial primordia. To our knowledge, a specific developmental arrest at this stage was not described before in A. nidulans. Initiation of the sexual cycle and differentiation processes leading to the general architecture of primordia are not impaired in the csn deletion strains.

But further differentiation and maturation of wall and ascospores is blocked. This suggests that after successful formation of the primordial structure, a regulatory process exists that is expendable for the first steps in sexual development leading to cleistothecial primordia but essential for completion of the sexual cycle. The COP9 signalosome seems to be an essential player in this regulatory process mediating maturation of primordia in A. nidulans. A similar developmental block at a level of metamorphosis of a primordial to mature stage can also be observed in homozygous CSN mutants of the fruit fly D. melangolaster. The mutant embryos hatch and develop normally until the middle of the third instar and frequently pupate, but then cease to develop and die (Freilich et al. 1999). This block in sexual

development seems to be the most severe phenotype of a defect in COP9 signalosome function in A. nidulans. And as stated above, the additional phenotypes of cell polarity and red pigmentation may also be related to developmental processes. It is thus conceivable that the COP9 signalosome in A. nidulans is dispensible for growth and housekeeping functions but essential for correct regulation of development.

Due to its impact on secondary metabolism, polarity and sexual development, the COP9 signalosome probably has several different downstream targets, summarised in Figure 4.10. This raises the question which upstream factors regulate CSN activity. An external signal important for development in A. nidulans is light, with the veA gene product as a proposed part of a corresponding signal transduction pathway. A csnD deletion strain is "blind" to light-regulation, like strains with constitutively low or high veA expression. Thus, the COP9 signalosome of A. nidulans is involved in light-dependent signalling and may even be connected with the same signal transduction pathway as VEA.

Notably, in the plant A. thaliana, the CSN is involved in the repression of photomorphogenesis in the dark. The proposed E3 ubiquitin ligase COP1 accumulates in the nucleus in the dark where it mediates, assisted among others by the COP9 signalosome, ubiquitinylation of an transcriptional activator of light-regulated genes (Osterlund et al. 1999; Osterlund et al. 2000; Schwechheimer and Deng 2001; Suzuki et al. 2002). The product of the A. nidulans veA gene has a negative influence on initiation on the asexual but a positive on the onset of the sexual cycle, as seen by the corresponding deletion and overproduction strains. It seems striking that the csnD deletion strain in a velvet wild-type background acts like a veA overproduction strain: a constitutive induction of the sexual cycle. In analogy to the findings in A. thaliana, it is thus conceivable that the COP9 signalosome of A. nidulans mediates a negative post-transcriptional effect on VEA, resulting in increased VEA protein levels in a csnD deletion strain. The function of the COP9 signalosome in light signalling might thus be genetically placed upstream or at the level of VEA, though this question should be addressed in future studies.

In summary, we present the first report of components of the COP9 signalosome in filamentous fungi and present strong evidence of its key regulatory function of development of the mold Aspergillus nidulans. The COP9 signalosome of A. nidulans is involved in several cellular processes including pigment synthesis, cell morphology, light-dependent signalling and specific sexual development. The function of the COP9 signalosome in filamentous fungi resembles in some respects that of higher eukaryotes. Because mutant strains are viable and can be propagated, this study represents an attractive basis to deliver new insights of the functions of the COP9 signalosome in eukaryotes.

4.6 References

Adams, T.H., J.K. Wieser, and J.H. Yu. 1998. Asexual sporulation in Aspergillus nidulans. Microbiol Mol Biol Rev 62: 35-54.

Altschul, S.F., W. Gish, W. Miller, E.W. Myers, and D.J. Lipman. 1990. Basic local alignment search tool. J Mol Biol 215: 403-410.

Axelrod, D.E., M. Gealt, and M. Pastushok. 1973. Gene control of developmental competence in Aspergillus nidulans. Dev Biol 34: 9-15.

Bech-Otschir, D., R. Kraft, X. Huang, P. Henklein, B. Kapelari, C. Pollmann, and W. Dubiel. 2001. COP9 signalosome-specific phosphorylation targets p53 to degradation by the ubiquitin system. EMBO J 20: 1630-1639.

Bech-Otschir, D., M. Seeger, and W. Dubiel. 2002. The COP9 signalosome: at the interface between signal transduction and ubiquitin-dependent proteolysis. J Cell Sci 115: 467-473.

Bennett, J.W. and L.L. Lasure. 1991. Growth media. In More gene manipulation in fungi (ed. J.W. Bennett and L.L. Lasure), pp. 441-457. Academic Press Inc, San Diego.

Bounpheng, M.A., I.N. Melnikova, S.G. Dodds, H. Chen, N.G. Copeland, D.J. Gilbert, N.A. Jenkins, and B.A.

Christy. 2000. Characterization of the mouse JAB1 cDNA and protein. Gene 242: 41-50.

Braus, G.H., S. Krappmann, and S.E. Eckert. 2002. Sexual development in ascomycetes: Fruit body formation of Aspergillus nidulans. In Molecular Biology of Fungal Development (ed. H.D. Osiewacz), pp. 215-244. Marcel Dekker, Inc., New Yok, Basel.

Busby, T.M., K.Y. Miller, and B.L. Miller. 1996. Suppression and enhancement of the Aspergillus nidulans medusa mutation by altered dosage of the bristle and stunted genes. Genetics 143: 155-163.

Busch, S., B. Hoffmann, O. Valerius, K. Starke, K. Duvel, and G.H. Braus. 2001. Regulation of the Aspergillus nidulans hisB gene by histidine starvation. Curr Genet 38: 314-22.

Bussink, H.J. and S.A. Osmani. 1998. A cyclin-dependent kinase family member (PHOA) is required to link developmental fate to environmental conditions in Aspergillus nidulans. EMBO J 17: 3990-4003.

Chamovitz, D.A., N. Wei, M.T. Osterlund, A.G. von Arnim, J.M. Staub, M. Matsui, and X.W. Deng. 1996. The COP9 complex, a novel multisubunit nuclear regulator involved in light control of a plant developmental switch. Cell 86: 115-121.

Champe, S.P. and A.A. el-Zayat. 1989. Isolation of a sexual sporulation hormone from Aspergillus nidulans. J Bacteriol 171: 3982-3988.

Champe, S.P., D.L. Nagle, and L.N. Yager. 1994. Sexual sporulation. In Aspergillus: 50 years on. Prog Ind Microbiol (ed. S.D. Martinelli and J.R. Klinghorn), pp. 429-454. Elsevier, Amsterdam.

Champe, S.P., P. Rao, and A. Chang. 1987. An endogenous inducer of sexual development in Aspergillus nidulans. J Gen Microbiol 133: 1383-1387.

Clutterbuck, A.J. 1969. A mutational analysis of conidial development in Aspergillus nidulans. Genetics 63:

317-327.

Clutterbuck, A.J. 1974. Aspergillus nidulans. In Handbook of Genetics. (ed. R.C. King), pp. 447-510. Plenum, New York.

Corpet, F. 1988. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16: 10881-10890.

Deng, X.W., W. Dubiel, N. Wei, K. Hofmann, K. Mundt, J. Colicelli, J. Kato, M. Naumann, D. Segal, M.

Seeger, A. Carr, M. Glickman, and D.A. Chamovitz. 2000. Unified nomenclature for the COP9 signalosome and its subunits: an essential regulator of development. Trends Genet 16: 202-203.

Eckert, S.E., B. Hoffmann, C. Wanke, and G.H. Braus. 1999. Sexual development of Aspergillus nidulans in

tryptophan auxotrophic strains. Arch Microbiol 172: 157-166.

ny.com/link/service/journals/00203/bibs/172n3p157.html.

Eckert, S.E., E. Kubler, B. Hoffmann, and G.H. Braus. 2000. The tryptophan synthase-encoding trpB gene of Aspergillus nidulans is regulated by the cross-pathway control system. Mol Gen Genet 263: 867-876.

Elble, R. 1992. A simple and efficient procedure for transformation of yeasts. Biotechniques 13: 18-20.

Fernandez-Abalos, J.M., H. Fox, C. Pitt, B. Wells, and J.H. Doonan. 1998. Plant-adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans. Mol Microbiol 27: 121-130.

Freilich, S., E. Oron, Y. Kapp, Y. Nevo-Caspi, S. Orgad, D. Segal, and D.A. Chamovitz. 1999. The COP9 signalosome is essential for development of Drosophila melanogaster. Curr Biol 9: 1187-1190.

Glickman, M.H., D.M. Rubin, O. Coux, I. Wefes, G. Pfeifer, Z. Cjeka, W. Baumeister, V.A. Fried, and D.

Finley. 1998. A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and eIF3. Cell 94: 615-623.

Golemis and R. Brent. 1996. Protein interaction studies. In Current protocols in molecular biology (ed. F.M.

Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidmann, A.J. Smith, and K. Struhl), pp. 429-454. Harvard Medical School, Massachusetts.

Guthrie, C. and G.R. Fink. 1991. Guide to yeast genetics and molecular biology. Methods Enzymol. 194: 15.

Gyuris, J., E. Golemis, H. Chertkov, and R. Brent. 1993. Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75: 791-803.

Han, D.M., Y.J. Han, K.S. Chae, K.Y. Jahng, and Y.H. Lee. 1994. Effects of various carbon sources on the development of Aspergillus nidulans with velA+ or velA1 allele. Korean. J. Mycol. 22: 332-337.

Han, K.H., K.Y. Han, J.H. Yu, K.S. Chae, K.Y. Jahng, and D.M. Han. 2001. The nsdD gene encodes a putative GATA-type transcription factor necessary for sexual development of Aspergillus nidulans.

Mol Microbiol 41: 299-309.

Hermann, T.E., M.B. Kurtz, and S.P. Champe. 1983. Laccase localized in hulle cells and cleistothecial primordia of Aspergillus nidulans. J Bacteriol 154: 955-964.

Hoffmann, B., C. Wanke, S.K. Lapaglia, and G.H. Braus. 2000. c-Jun and RACK1 homologues regulate a control point for sexual development in Aspergillus nidulans. Mol Microbiol 37: 28-41.

Howard, B.H. and H. Raistrick. 1955. The colouring matters of species in the Aspergillus nidulans group.

Biochem. J. 59: 476-484.

Inoue, H., H. Nojima, and H. Okayama. 1990. High efficiency transformation of Escherichia coli with plasmids.

Gene 96: 23-28.

Käfer, E. 1977. Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations. Adv Genet 19: 33-131.

Kapelari, B., D. Bech-Otschir, R. Hegerl, R. Schade, R. Dumdey, and W. Dubiel. 2000. Electron microscopy and subunit-subunit interaction studies reveal a first architecture of COP9 signalosome. J Mol Biol 300: 1169-1178.

Kim, T., K. Hofmann, A.G. von Arnim, and D.A. Chamovitz. 2001. PCI complexes: pretty complex interactions in diverse signaling pathways. Trends Plant Sci 6: 379-386.

Kirk, K.E. and N.R. Morris. 1991. The tubB alpha-tubulin gene is essential for sexual development in Aspergillus nidulans. Genes Dev 5: 2014-2023.

Kwok, S.F., R. Solano, T. Tsuge, D.A. Chamovitz, J.R. Ecker, M. Matsui, and X.W. Deng. 1998. Arabidopsis homologs of a c-Jun coactivator are present both in monomeric form and in the COP9 complex, and their abundance is differentially affected by the pleiotropic cop/det/fus mutations. Plant Cell 10:

1779-1790.

Lee, B.S. and J.W. Taylor. 1990. Isolation of DNA from fungal mycelia and single spores. In PCR protocols: A

Lee, B.S. and J.W. Taylor. 1990. Isolation of DNA from fungal mycelia and single spores. In PCR protocols: A

Im Dokument Amino Acid Biosynthesis and the COP9 Signalosome in Aspergillus nidulans (Seite 97-0)